Original Article
Clinical significance of hepatitis B virion and SVP productivity: relationships between intrahepatic and serum markers in chronic hepatitis B patients
United European Gastroenterology Journal 2014, Vol. 2(2) 99–107 ! Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/2050640614525151 ueg.sagepub.com
Cosmas Rinaldi Adithya Lesmana1,4, Kathy Jackson2, Seng Gee Lim3, Ali Sulaiman1, Levina S Pakasi4, Rino A Gani1, Irsan Hasan1, Andri Sanityoso Sulaiman1, Laurentius A Lesmana1,4, Rachel Hammond2, Peter Revill2, Stephen Locarnini2 and Scott David Bowden2
Abstract Background: Clinical use of hepatitis B viral (HBV) quantitative seromarker\s remains questionable since it is not precisely known whether they represent intrahepatic viral replication. Covalently closed circular DNA (cccDNA), relaxed circular DNA (rcDNA), and pregenomic RNA (pgRNA) are more likely to represent active HBV replication and their measurement can be used to derive virion productivity (VP; rcDNA/cccDNA), subviral particle (SVP) productivity (quantitative HBsAg/cccDNA), and replicative activity (RA; pgRNA/cccDNA). These can be used to compare relative HBV replication between HBeAg-negative and -positive patients. Objective: To study the clinical significance of intrahepatic HBV replication phenomenon between HBeAg-negative and -positive patients and its correlation with quantitative HBV seromarkers. Method: This was a prospective study between January 2010 and December 2011. Study subjects were naive chronic hepatitis B patients from Cipto Mangunkusumo and Medistra Hospitals. All patient samples underwent liver biochemistry and HBV seromarkers testing (HBeAg, quantitative HBsAg and HBV DNA levels), and patients underwent liver biopsy. Stored liver specimens were analysed for intrahepatic rcDNA, cccDNA, and pgRNA with quantification performed by real-time PCR. Comparison of HBV markers between HBsAg-positive and -negative patients was carried out using the Mann–Whitney U-test. Pearson’s correlation test was performed among HBV intrahepatic and seromarkers using their log-transformed values. Results: A total of 104 patients were enrolled in this study; 54 (51.9%) were male. Patients’ mean age was 41.9 11.63 years (range 19–70 years). Sixty-one patients (58.7%) were HBeAg-negative. All HBV markers were significantly higher in HBeAgpositive than HBeAg-negative patients, except for SVP productivity and RA. Serum HBV DNA was strongly correlated with intrahepatic total HBV DNA (r ¼ 0.771), cccDNA (r ¼ 0.774), and rcDNA (r ¼ 0.780) while serum quantitative HBsAg showed only moderate correlation with intrahepatic total DNA (r ¼ 0.671), cccDNA (r ¼ 0.632), rcDNA (r ¼ 0.675), and SVP productivity (r ¼ 0.557). Conclusions: Serum HBV DNA concentration and quantitative HBsAg might not accurately predict intrahepatic viral activity. Virion and SVP production do not occur in parallel with replicative activity.
Keywords Chronic hepatitis B, intrahepatic cccDNA, quantitative HBsAg, subviral particles productivity, total HBV DNA, virion productivity Received: 24 September 2013; accepted: 30 January 2014 4
Digestive Disease and GI Oncology Center, Medistra Hospital, Jakarta, Indonesia
1
Hepatology, Medicine, University of Indonesia, Cipto Mangunkusumo Hospital, Jakarta, Indonesia 2 Victorian Infectious Disease Reference Laboratory, Melbourne, Australia 3 Division of Gastroenterology, Medicine, National University Hospital, Singapore, Singapore
Corresponding author: Cosmas Rinaldi Adithya Lesmana, Hepatobiliary, Internal Medicine, Cipto Mangunkusumo Hospital, Jl. Diponegoro 71, Jakarta, DKI Jakarta 10430, Indonesia. Email: [email protected]
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United European Gastroenterology Journal 2(2)
Introduction Treatment of chronic hepatitis B (CHB) infection relies upon monitoring serological markers (HBsAg, HBeAg) and HBV DNA levels.1–3 Decreases in HBV DNA levels are used as an indicator of efficacious antiviral therapy,4 whereas HBsAg loss is considered the ultimate goal of serological viral response. In patients with positive HBeAg, HBeAg seroconversion is a desirable treatment endpoint. Complete eradication of HBV by antiviral therapy is still difficult to achieve because of the persistence of viral covalently closed circular DNA (cccDNA) in the hepatocyte nucleus.5,6 This HBV replicative form is produced from the partially double-stranded genomic DNA (relaxed circular DNA, rcDNA) in the virion, which after infection, is converted by host cell enzymes in the nucleus into a fully double-stranded form. From this, HBV cccDNA molecule is generated as the transcriptional template for HBV RNA production. The hepatocyte RNA polymerase II transcribes all the viral mRNA from the HBV cccDNA, including the HBV pregenomic RNA (pgRNA).7 The pgRNA functions as the mRNA not only for the polymerase and core protein production, but it can also be reverse transcribed after encapsidation into new genomic rcDNA. Capsids containing the newly formed genomic DNA can be enveloped by surface proteins and secreted into the blood or redirected back to the nucleus to maintain a pool of HBV cccDNA.6 Therefore, the measurement of pgRNA provides more accurate information on viral replication and the ratio of pgRNA per cccDNA molecule reflect the true viral capacity to replicate its genomic material. This ratio has been termed as replicative activity (RA). Presuming in established CHB infection that most of the intrahepatic rcDNA is aimed for virion production, it has been proposed also that the ratio of rcDNA per cccDNA molecule reflects virion productivity (VP).8 Since not all new rcDNA will be part of the new intact virion, neither rcDNA nor RA can be used to reflect the capacity of viral particle production. Virion productivity may thus be a measure of the actual production of viral particles. Recently, quantitative HBsAg has been introduced to help predict treatment response in CHB patients. This marker is believed to better reflect intrahepatic viral replication than serum HBV DNA levels.9,10 Indeed, HBsAg titres have been proposed as a surrogate marker for transcriptionally active HBV cccDNA, and in a recent study of treatment-naive patients with CHB, an association between HBsAg titres, cccDNA, and total intrahepatic HBV DNA was shown but only in HBeAg-positive patients.11 However, commercial
assays to quantify HBsAg detect all forms of surface antigen in the blood, either as an intact viral particle (virion) or as spherical and filamentous subviral particles (SVP).12,13 Conflicting results have been published among studies conducted in different countries on the use of quantitative HBsAg in clinical practice and its exact role is yet to be determined.14,15 The lack of association in HBeAg-negative patients indicates that the association of HBsAg titres with intrahepatic HBV DNA is not straightforward and is likely affected by both virus and host factors. The different natural history of CHB infection and HBV genotypes between Western and Asian countries, and even between Asian countries themselves might be one of the reasons for the variation in results in clinical studies.16,17 Molecular studies on intrahepatic HBV DNA kinetics may give a new perspective in understanding HBV replication and CHB, distinct from the biomarkers that are usually monitored in clinical practice. This study was aimed at evaluating the clinical significance of virion and SVP productivity by measuring intrahepatic HBV DNA replicative forms and serological markers in HBeAg-positive and HBeAg-negative patients.
Methods Study design and subjects This was a prospective cross-sectional study conducted in the Department of Internal Medicine, University of Indonesia from January 2010 to December 2011. All consecutive, treatment-naı¨ ve, newly diagnosed CHB patients from two big referral hospitals, Cipto Mangunkusumo and Medistra Hospitals, Jakarta who presented to the outpatient clinics and agreed to participate in the study after signing informed consent were enrolled. This study was approved by the Ethical Committee for Medical Research, Faculty of Medicine, University of Indonesia. Chronic hepatitis B infection was diagnosed by the presence of HBsAg in the patient’s serum at least for 6 months based on a chemiluminescent microparticle immunoassay (CMIA; ARCHITECT HBsAg Reagent Kit; Abbott Diagnostics, Abbott Park, IL, USA). Inclusion criteria were newly diagnosed, treatmentnaive CHB patients, who were willing to undergo liver biopsy before and after antiviral treatment. Patients were excluded if there was a coinfection with hepatitis C virus or human immunodeficiency virus, decompensated liver cirrhosis, or hepatocellular carcinoma. Quantitative serum HBsAg measurement, HBV DNA levels, and genotyping were done in a private
Downloaded from ueg.sagepub.com at SETON HALL UNIV on March 30, 2015
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laboratory in Jakarta, whereas measurement of intrahepatic markers (cccDNA and pgRNA assay) and identification of the PC/BCP variants were done in Victorian Infectious Disease Reference Laboratory, Melbourne, Australia.
biopsy stored at 80 C or in an RNA stabilization solution at 20 C (RNALater; Ambion, Austin, Texas, USA) for analysis, including both cccDNA and pgRNA quantification. Fibrosis stage was assessed using the METAVIR scoring system.19,20
Quantitative HBV seromarker measurement
Intrahepatic total and cccDNA quantification
Quantitative HBsAg was measured using CMIA. The results were expressed as signal sample/cutoff ratios (S/ CO). The values were then calibrated (ARCHITECT HBsAg Calibrators) and verified (ARCHITECT HBsAg Controls) to obtain quantitative levels (IU/ml). Manual dilution was performed if the HBsAg value exceeding 250 IU/ml. The suggested dilution was 1:500, which was done by adding 25 ml specimens to 475 l ARCHITECT HBsAg Manual Diluent for 1:20 dilution, followed by adding 20 ml of the first solution to 480 l ARCHITECT HBsAg Manual Diluent for a 1:500 dilution. According to the manufacturer, the standard error was 0.018 and the coefficient of variation was 7.8%. Specimens were not analysed in duplicate. Serum HBV DNA level was measured with COBAS TaqMan HBV Test (Roche Diagnostics, Mannheim, Germany). The detection range was 30 IU/ml to 1 108 IU/ml. Samples were diluted and reanalysed if the titre was above the upper limit of quantification. Serum samples were obtained in the same day of liver biopsy.
Determination of HBV cccDNA levels was carried out by real-time PCR using a LightCycler 1.0 and LightCycler version 3.5 software (Roche Diagnostics). Reaction volumes (20 ml) consisted of 2 ml extracted DNA, 5 mM MgCl2, 0.5 mM primer mix and 0.2 mM hybridization probes. Selective HBV cccDNA primers consisted of two upstream primers (CCC1 50 GCGGWCTCCCCGTCTGTGCC-30 and CCC3 50 -GTCTGTGCCTTCTCATCTGC-30 ) and a downstream primer (CCC2 50 -GTCCATGCCCCAAA GCCACC-30 ). FRET hybridization probes were 50 GTTCACGGTGGTCTCCATGCGACGT-FL-30 and 50 -LCR640-AGGTGAAGCGAAGTGCACACGGWC C-30 . Total intrahepatic DNA was measured using primers RC1 50 -CTCGTGGTGGACTTCTCTC-30 and RC2 50 -CAGCAGGATGAAGAGGAA-30 and FRET probes 50 - CACTCACCAACCTSYTGTCCTCCAAFL-30 and 50 ¼ LCR640-TGTCCTGGYTATCGCT GGATGTGTCT-30 . Quantification standards were derived by dilution of a linearized plasmid (pHBV EcoR1) containing a greater than full-length HBV genome of genotype D. This standard has been previously titrated against the WHO international HBV reference standard to correlate quantification values. Variation in the amounts of liver tissue was normalized by quantifying b-globin in each sample with the Roche DNA control kit. The relaxed circular HBV DNA was calculated as total HBV DNA – cccDNA.
Identification of HBV genotype and precore and basal core promoter variants Hepatitis B virus genotyping was initially determined using line probe assay (INNO-LiPA HBV; Innogenetics, Belgium). HBV genotype was confirmed by sequence analysis of the HBV surface/polymerase gene as previously described.18 Variants with substitutions in the precore (PC) region and basal core promoter (BCP) region were identified by sequencing after PCR amplification, as described by Ayres et al.18 The PC variant was defined by the presence of the G1896A mutation and the BCP variant by the A1762T/G1764A mutation.
Liver biopsy Liver biopsy was performed on all patients and histopathological assessment was carried out by an experienced liver pathologist who was blinded to the clinical data. Ultrasound-guided biopsy was performed using a 16-gauge Menghini needle (Hepafix; B Braun Melsungen, Melsungen, Germany) under local anaesthesia. Tissue specimens were first prepared for histopathology examination and the other portions of the
Intrahepatic pregenomic (Pg) RNA measurement The RNA extraction was performed using RNeasy Mini Kit (Qiagen, Germany). Fresh frozen liver tissues that were previously stored in RNALater tubes were disrupted by homogenization and the lysate was purified according to the manufacturer’s instructions. A further purification of RNA was carried out using MicroPoly(A) Purist kit (Life Technologies, CA, USA) to remove any DNA carryover. Levels of hepatic RNA were normalized using the commercially available human G6PDH Housekeeping Gene Set (Roche Diagnostics). After reverse transcription of RNA to cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, USA), amplification and quantification of pgRNA was as previously described.21
Downloaded from ueg.sagepub.com at SETON HALL UNIV on March 30, 2015
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United European Gastroenterology Journal 2(2)
Replicative activity, virion productivity, and subviral particle productivity As well as quantification of HBV seromarkers and intrahepatic markers, there were several ratios derived from these markers. Replicative activity was a ratio of pgRNA production per cccDNA molecule (pgRNA/ cccDNA). Virion productivity was calculated as rcDNA levels per cccDNA molecule (rcDNA/ cccDNA). Subviral particle productivity was the quantitative serum HBsAg levels divided by cccDNA levels (qHBsAg/cccDNA).
Statistical analysis Characteristics of the study subjects were presented descriptively as proportion or median. Comparison between HBeAg-positive and -negative were tested using the chi-squared test for categorical data and the Mann–Whitney U-test for skewed numerical data. Mean differences of log-transformed values were calculated using t-test. Pearson’s correlation test was applied on the log-transformed values of intrahepatic and serum markers. A p-value
Clinical significance of hepatitis B virion and SVP productivity: relationships between intrahepatic and serum markers in chronic hepatitis B patients
United European Gastroenterology Journal 2014, Vol. 2(2) 99–107 ! Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/2050640614525151 ueg.sagepub.com
Cosmas Rinaldi Adithya Lesmana1,4, Kathy Jackson2, Seng Gee Lim3, Ali Sulaiman1, Levina S Pakasi4, Rino A Gani1, Irsan Hasan1, Andri Sanityoso Sulaiman1, Laurentius A Lesmana1,4, Rachel Hammond2, Peter Revill2, Stephen Locarnini2 and Scott David Bowden2
Abstract Background: Clinical use of hepatitis B viral (HBV) quantitative seromarker\s remains questionable since it is not precisely known whether they represent intrahepatic viral replication. Covalently closed circular DNA (cccDNA), relaxed circular DNA (rcDNA), and pregenomic RNA (pgRNA) are more likely to represent active HBV replication and their measurement can be used to derive virion productivity (VP; rcDNA/cccDNA), subviral particle (SVP) productivity (quantitative HBsAg/cccDNA), and replicative activity (RA; pgRNA/cccDNA). These can be used to compare relative HBV replication between HBeAg-negative and -positive patients. Objective: To study the clinical significance of intrahepatic HBV replication phenomenon between HBeAg-negative and -positive patients and its correlation with quantitative HBV seromarkers. Method: This was a prospective study between January 2010 and December 2011. Study subjects were naive chronic hepatitis B patients from Cipto Mangunkusumo and Medistra Hospitals. All patient samples underwent liver biochemistry and HBV seromarkers testing (HBeAg, quantitative HBsAg and HBV DNA levels), and patients underwent liver biopsy. Stored liver specimens were analysed for intrahepatic rcDNA, cccDNA, and pgRNA with quantification performed by real-time PCR. Comparison of HBV markers between HBsAg-positive and -negative patients was carried out using the Mann–Whitney U-test. Pearson’s correlation test was performed among HBV intrahepatic and seromarkers using their log-transformed values. Results: A total of 104 patients were enrolled in this study; 54 (51.9%) were male. Patients’ mean age was 41.9 11.63 years (range 19–70 years). Sixty-one patients (58.7%) were HBeAg-negative. All HBV markers were significantly higher in HBeAgpositive than HBeAg-negative patients, except for SVP productivity and RA. Serum HBV DNA was strongly correlated with intrahepatic total HBV DNA (r ¼ 0.771), cccDNA (r ¼ 0.774), and rcDNA (r ¼ 0.780) while serum quantitative HBsAg showed only moderate correlation with intrahepatic total DNA (r ¼ 0.671), cccDNA (r ¼ 0.632), rcDNA (r ¼ 0.675), and SVP productivity (r ¼ 0.557). Conclusions: Serum HBV DNA concentration and quantitative HBsAg might not accurately predict intrahepatic viral activity. Virion and SVP production do not occur in parallel with replicative activity.
Keywords Chronic hepatitis B, intrahepatic cccDNA, quantitative HBsAg, subviral particles productivity, total HBV DNA, virion productivity Received: 24 September 2013; accepted: 30 January 2014 4
Digestive Disease and GI Oncology Center, Medistra Hospital, Jakarta, Indonesia
1
Hepatology, Medicine, University of Indonesia, Cipto Mangunkusumo Hospital, Jakarta, Indonesia 2 Victorian Infectious Disease Reference Laboratory, Melbourne, Australia 3 Division of Gastroenterology, Medicine, National University Hospital, Singapore, Singapore
Corresponding author: Cosmas Rinaldi Adithya Lesmana, Hepatobiliary, Internal Medicine, Cipto Mangunkusumo Hospital, Jl. Diponegoro 71, Jakarta, DKI Jakarta 10430, Indonesia. Email: [email protected]
Downloaded from ueg.sagepub.com at SETON HALL UNIV on March 30, 2015
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United European Gastroenterology Journal 2(2)
Introduction Treatment of chronic hepatitis B (CHB) infection relies upon monitoring serological markers (HBsAg, HBeAg) and HBV DNA levels.1–3 Decreases in HBV DNA levels are used as an indicator of efficacious antiviral therapy,4 whereas HBsAg loss is considered the ultimate goal of serological viral response. In patients with positive HBeAg, HBeAg seroconversion is a desirable treatment endpoint. Complete eradication of HBV by antiviral therapy is still difficult to achieve because of the persistence of viral covalently closed circular DNA (cccDNA) in the hepatocyte nucleus.5,6 This HBV replicative form is produced from the partially double-stranded genomic DNA (relaxed circular DNA, rcDNA) in the virion, which after infection, is converted by host cell enzymes in the nucleus into a fully double-stranded form. From this, HBV cccDNA molecule is generated as the transcriptional template for HBV RNA production. The hepatocyte RNA polymerase II transcribes all the viral mRNA from the HBV cccDNA, including the HBV pregenomic RNA (pgRNA).7 The pgRNA functions as the mRNA not only for the polymerase and core protein production, but it can also be reverse transcribed after encapsidation into new genomic rcDNA. Capsids containing the newly formed genomic DNA can be enveloped by surface proteins and secreted into the blood or redirected back to the nucleus to maintain a pool of HBV cccDNA.6 Therefore, the measurement of pgRNA provides more accurate information on viral replication and the ratio of pgRNA per cccDNA molecule reflect the true viral capacity to replicate its genomic material. This ratio has been termed as replicative activity (RA). Presuming in established CHB infection that most of the intrahepatic rcDNA is aimed for virion production, it has been proposed also that the ratio of rcDNA per cccDNA molecule reflects virion productivity (VP).8 Since not all new rcDNA will be part of the new intact virion, neither rcDNA nor RA can be used to reflect the capacity of viral particle production. Virion productivity may thus be a measure of the actual production of viral particles. Recently, quantitative HBsAg has been introduced to help predict treatment response in CHB patients. This marker is believed to better reflect intrahepatic viral replication than serum HBV DNA levels.9,10 Indeed, HBsAg titres have been proposed as a surrogate marker for transcriptionally active HBV cccDNA, and in a recent study of treatment-naive patients with CHB, an association between HBsAg titres, cccDNA, and total intrahepatic HBV DNA was shown but only in HBeAg-positive patients.11 However, commercial
assays to quantify HBsAg detect all forms of surface antigen in the blood, either as an intact viral particle (virion) or as spherical and filamentous subviral particles (SVP).12,13 Conflicting results have been published among studies conducted in different countries on the use of quantitative HBsAg in clinical practice and its exact role is yet to be determined.14,15 The lack of association in HBeAg-negative patients indicates that the association of HBsAg titres with intrahepatic HBV DNA is not straightforward and is likely affected by both virus and host factors. The different natural history of CHB infection and HBV genotypes between Western and Asian countries, and even between Asian countries themselves might be one of the reasons for the variation in results in clinical studies.16,17 Molecular studies on intrahepatic HBV DNA kinetics may give a new perspective in understanding HBV replication and CHB, distinct from the biomarkers that are usually monitored in clinical practice. This study was aimed at evaluating the clinical significance of virion and SVP productivity by measuring intrahepatic HBV DNA replicative forms and serological markers in HBeAg-positive and HBeAg-negative patients.
Methods Study design and subjects This was a prospective cross-sectional study conducted in the Department of Internal Medicine, University of Indonesia from January 2010 to December 2011. All consecutive, treatment-naı¨ ve, newly diagnosed CHB patients from two big referral hospitals, Cipto Mangunkusumo and Medistra Hospitals, Jakarta who presented to the outpatient clinics and agreed to participate in the study after signing informed consent were enrolled. This study was approved by the Ethical Committee for Medical Research, Faculty of Medicine, University of Indonesia. Chronic hepatitis B infection was diagnosed by the presence of HBsAg in the patient’s serum at least for 6 months based on a chemiluminescent microparticle immunoassay (CMIA; ARCHITECT HBsAg Reagent Kit; Abbott Diagnostics, Abbott Park, IL, USA). Inclusion criteria were newly diagnosed, treatmentnaive CHB patients, who were willing to undergo liver biopsy before and after antiviral treatment. Patients were excluded if there was a coinfection with hepatitis C virus or human immunodeficiency virus, decompensated liver cirrhosis, or hepatocellular carcinoma. Quantitative serum HBsAg measurement, HBV DNA levels, and genotyping were done in a private
Downloaded from ueg.sagepub.com at SETON HALL UNIV on March 30, 2015
Lesmana et al.
101
laboratory in Jakarta, whereas measurement of intrahepatic markers (cccDNA and pgRNA assay) and identification of the PC/BCP variants were done in Victorian Infectious Disease Reference Laboratory, Melbourne, Australia.
biopsy stored at 80 C or in an RNA stabilization solution at 20 C (RNALater; Ambion, Austin, Texas, USA) for analysis, including both cccDNA and pgRNA quantification. Fibrosis stage was assessed using the METAVIR scoring system.19,20
Quantitative HBV seromarker measurement
Intrahepatic total and cccDNA quantification
Quantitative HBsAg was measured using CMIA. The results were expressed as signal sample/cutoff ratios (S/ CO). The values were then calibrated (ARCHITECT HBsAg Calibrators) and verified (ARCHITECT HBsAg Controls) to obtain quantitative levels (IU/ml). Manual dilution was performed if the HBsAg value exceeding 250 IU/ml. The suggested dilution was 1:500, which was done by adding 25 ml specimens to 475 l ARCHITECT HBsAg Manual Diluent for 1:20 dilution, followed by adding 20 ml of the first solution to 480 l ARCHITECT HBsAg Manual Diluent for a 1:500 dilution. According to the manufacturer, the standard error was 0.018 and the coefficient of variation was 7.8%. Specimens were not analysed in duplicate. Serum HBV DNA level was measured with COBAS TaqMan HBV Test (Roche Diagnostics, Mannheim, Germany). The detection range was 30 IU/ml to 1 108 IU/ml. Samples were diluted and reanalysed if the titre was above the upper limit of quantification. Serum samples were obtained in the same day of liver biopsy.
Determination of HBV cccDNA levels was carried out by real-time PCR using a LightCycler 1.0 and LightCycler version 3.5 software (Roche Diagnostics). Reaction volumes (20 ml) consisted of 2 ml extracted DNA, 5 mM MgCl2, 0.5 mM primer mix and 0.2 mM hybridization probes. Selective HBV cccDNA primers consisted of two upstream primers (CCC1 50 GCGGWCTCCCCGTCTGTGCC-30 and CCC3 50 -GTCTGTGCCTTCTCATCTGC-30 ) and a downstream primer (CCC2 50 -GTCCATGCCCCAAA GCCACC-30 ). FRET hybridization probes were 50 GTTCACGGTGGTCTCCATGCGACGT-FL-30 and 50 -LCR640-AGGTGAAGCGAAGTGCACACGGWC C-30 . Total intrahepatic DNA was measured using primers RC1 50 -CTCGTGGTGGACTTCTCTC-30 and RC2 50 -CAGCAGGATGAAGAGGAA-30 and FRET probes 50 - CACTCACCAACCTSYTGTCCTCCAAFL-30 and 50 ¼ LCR640-TGTCCTGGYTATCGCT GGATGTGTCT-30 . Quantification standards were derived by dilution of a linearized plasmid (pHBV EcoR1) containing a greater than full-length HBV genome of genotype D. This standard has been previously titrated against the WHO international HBV reference standard to correlate quantification values. Variation in the amounts of liver tissue was normalized by quantifying b-globin in each sample with the Roche DNA control kit. The relaxed circular HBV DNA was calculated as total HBV DNA – cccDNA.
Identification of HBV genotype and precore and basal core promoter variants Hepatitis B virus genotyping was initially determined using line probe assay (INNO-LiPA HBV; Innogenetics, Belgium). HBV genotype was confirmed by sequence analysis of the HBV surface/polymerase gene as previously described.18 Variants with substitutions in the precore (PC) region and basal core promoter (BCP) region were identified by sequencing after PCR amplification, as described by Ayres et al.18 The PC variant was defined by the presence of the G1896A mutation and the BCP variant by the A1762T/G1764A mutation.
Liver biopsy Liver biopsy was performed on all patients and histopathological assessment was carried out by an experienced liver pathologist who was blinded to the clinical data. Ultrasound-guided biopsy was performed using a 16-gauge Menghini needle (Hepafix; B Braun Melsungen, Melsungen, Germany) under local anaesthesia. Tissue specimens were first prepared for histopathology examination and the other portions of the
Intrahepatic pregenomic (Pg) RNA measurement The RNA extraction was performed using RNeasy Mini Kit (Qiagen, Germany). Fresh frozen liver tissues that were previously stored in RNALater tubes were disrupted by homogenization and the lysate was purified according to the manufacturer’s instructions. A further purification of RNA was carried out using MicroPoly(A) Purist kit (Life Technologies, CA, USA) to remove any DNA carryover. Levels of hepatic RNA were normalized using the commercially available human G6PDH Housekeeping Gene Set (Roche Diagnostics). After reverse transcription of RNA to cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, USA), amplification and quantification of pgRNA was as previously described.21
Downloaded from ueg.sagepub.com at SETON HALL UNIV on March 30, 2015
102
United European Gastroenterology Journal 2(2)
Replicative activity, virion productivity, and subviral particle productivity As well as quantification of HBV seromarkers and intrahepatic markers, there were several ratios derived from these markers. Replicative activity was a ratio of pgRNA production per cccDNA molecule (pgRNA/ cccDNA). Virion productivity was calculated as rcDNA levels per cccDNA molecule (rcDNA/ cccDNA). Subviral particle productivity was the quantitative serum HBsAg levels divided by cccDNA levels (qHBsAg/cccDNA).
Statistical analysis Characteristics of the study subjects were presented descriptively as proportion or median. Comparison between HBeAg-positive and -negative were tested using the chi-squared test for categorical data and the Mann–Whitney U-test for skewed numerical data. Mean differences of log-transformed values were calculated using t-test. Pearson’s correlation test was applied on the log-transformed values of intrahepatic and serum markers. A p-value
